Interleukin 28 Is a Potential Therapeutic Target for Sepsis

Clinical Immunology 205 (2019) 29–34

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Clinical Immunology

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Interleukin 28 is a potential therapeutic target for sepsis T ⁎ Qin Luoa,b, Yi Liuc, Shuang Liub, Yibing Yinb, Banglao Xud, Ju Caoa, a Department of Laboratory Medicine, The First Aﬃliated Hospital of Chongqing Medical University, Chongqing, China b Key Laboratory of Diagnostic Medicine designated by the Ministry of Education, Chongqing Medical University, Chongqing, China c Department of Intensive Care Unit, The Second Aﬃliated Hospital of Chongqing Medical University, Chongqing, China d Department of Laboratory Medicine, Guangzhou First People's Hospital, School of Medicine, South China University of Technology, Guangzhou, Guangdong, China

ARTICLE INFO ABSTRACT

Keywords: Identification of new therapeutic targets for the treatment of sepsis is imperative. We report here that cytokine Interleukin-28 IL-28 (IFN-λ) levels were elevated in clinical and experimental sepsis. Neutralization of IL-28 protected mice Sepsis from lethal sepsis induced by cecal ligation and puncture (CLP), which was associated with improved bacterial Infection clearance and enhanced neutrophil infiltration. Conversely, administration of recombinant IL-28 aggravated Immunity mortality, facilitated bacterial dissimilation and limited neutrophil recruitment, in the model of sepsis induced Neutrophil by CLP. This study defines IL-28 as a detrimental mediator during sepsis and identifies a potential therapeutic target for the immune therapy in sepsis.

1. Introduction immunopathology of sepsis is still poorly understood. To address this issue, we examined the potential role of IL-28 in the Each year, about 31.5 million individuals develop sepsis, and up to progression of sepsis. Blood specimens from patients with sepsis de- 5.3 million deaths due to sepsis occur worldwide [1]. The current monstrated increased release of IL-28A. We used the cecal ligation and treatment of sepsis relies on the administration of antibiotics and organ puncture (CLP) model of sepsis to test effects of IL-28 on sepsis. function support, and there is no specific therapeutic agent approved Neutralization of IL-28 reduced CLP-induced sepsis mortality, while IL- for the treatment of sepsis [2]. In sepsis, the host immune response 28 administration in sepsis increased mortality. This study identifies triggered by an invading pathogen fails to return to homeostasis, re- cytokine IL-28 as a new immunotherapeutic agent for the treatment of sulting in aberrant inflammation and immune suppression, and septic sepsis. patients fail to eradicate primary infections and are susceptible to secondary, mostly opportunistic, infections [3]. Identification of the 2. Materials and methods immune factors involved in sepsis-induced aberrant inflammation and immune suppression will provide novel potential targets for in- 2.1. Study subjects and data collection dividualized immune therapy in the patients with sepsis. The classical IFN family cytokines, such as types I and II IFNs, have Patients who met the clinical criteria for sepsis-3 were screened for been shown to play an important role in regulating host immune re- eligibility within the first 24 h after they were admitted to the Intensive sponses during sepsis [4]. Type III IFNs, or IFN-λs, are a newly de- Care Unit of The Second Affiliated Hospital of Chongqing Medical scribed member of the IFN family, and they consist of three members in University between September 2015 and December 2017 [8]. A total of humans, denoted IL-28A (IFN-λ2), IL-28B (IFN-λ3), and IL-29 (IFN-λ1), 46 septic patients were enrolled. Patient data such as Acute Physiology and two members in mice (IL-28A and IL-28B) [4]. IFN-λs signal and Chronic Health Evaluation II (APACHE II) score, Sequential Organ through a heterodimeric receptor consisting of the IL-28 receptor (IL- Failure Assessment (SOFA) score, the counts of white blood cells (WBC) 28R) and IL-10RB, which is expressed predominantly on mucosal sur- and the levels of C-reaction proteins (CRP), microbial culture results, faces and on neutrophils [5,6]. IL-28 cytokine family members could be the length of ICU stay and hospital stay, and the outcome of ICU stay produced by various cells upon viral infection or Toll like receptor li- were recorded. Patients with malignancy, organ transplantation, HIV- gation [7]. Although the antiviral and anti-tumor activity has been infected patients, and patients receiving immunosuppressive agents in extensively studied during the past decade, the role of IL-28 in the the past 8 weeks were excluded from the study. 26 healthy donors with

⁎ Corresponding author at: Department of Laboratory Medicine, The First Aﬃliated Hospital of Chongqing Medical University, Yuzhong District, Chongqing, China. E-mail address: [email protected] (J. Cao). https://doi.org/10.1016/j.clim.2019.05.012 Received 23 April 2019; Received in revised form 17 May 2019; Accepted 17 May 2019 Available online 20 May 2019 1521-6616/ © 2019 Elsevier Inc. All rights reserved. Q. Luo, et al. Clinical Immunology 205 (2019) 29–34

Table 1 no medical problems in the medical examination center of The First Baseline characteristics of patients with sepsis and healthy controls. Aﬃliated Hospital of Chongqing Medical University were also included

Characteristics Sepsis patients Healthy controls as controls. This protocol was approved by the Clinical Research Ethics (n = 46) (n = 26) Committee of Chongqing Medical University, and informed consent was obtained from all participants according to the Declaration of Helsinki. Male sex 22 11 Age, years 55 (45–71) 49 (40–65) 9 WBC, 10 /L 9 (6–19) 6 (4–9) 2.2. Sepsis model CRP, mg/L 120 (30–216) NA Infection site, no. of patients Respiratory 17 NA Cecal ligation puncture (CLP) was used as a model of sepsis as de- Abdominal 16 NA scribed in our previous studies [9,10]. Briefly, C57BL/6 mice were Vascular 3 NA anesthetized intraperitoneally (i.p.) with a mixture of xylazine (4.5 mg/ Urinary 5 NA kg) and ketamine (90 mg/kg), and the cecum was exposed, ligatured at Other 5 NA its external third, and punctured through with a 21-gauge needle. The Bacteremia 30 NA Isolates, no. of patients cecum was then returned to the peritoneal cavity, and incisions were Gram positive 12 NA closed. Sham-operated (control) animals underwent identical lapar- Gram negative 22 NA atomy, and the cecum was exposed but not ligated or punctured and Fungus 1 NA was then replaced in the peritoneal cavity. Mice received saline (5 ml Miscellaneous 6 NA Unknown 5 NA per 100 g body weight) subcutaneously for resuscitation. Survival was APACHE II score 16.2 (13.1–22.5) NA monitored twice daily for 14 d. All experiments involving animals ad- SOFA score 8.2 (5.1–15.5) NA hered to guidelines and received the approval of the Institutional Re- – ICU stay, days 11 (3 19) NA view Committee for Animal Care and Use at Chongqing Medical Uni- Died/survived 6/40 NA versity. NOTE. Data are expressed as median (interquartile range) unless otherwise indicated. APACHE II: acute physiology and chronic health evaluation II; SOFA: 2.3. Measurement of IL-28 sequential organ failure assessment; ICU: intensive care unit; WBC: white blood cells; CRP: C-reaction protein; NA: not applicable. IL-28 cytokines were quantified using specific ELISA kits, following the manufacturer's instructions. Mouse IL-28A/B kits were from R&D Systems, and human IL-28A (cross-reacting with IL-28B) kits were also from R&D Systems.

Fig. 1. IL-28 protein levels were elevated in clinical and experimental sepsis (A) IL-28A concentrations were measured by ELISA in serum samples collected from 46 patients with sepsis (40 survivors and 6 non-survivors) and from 26 healthy control subjects. Horizontal bars represent median values, and dots represent individual participants. *p < .05, compared between groups (denoted by horizontal bracket; Kruskal-Wallis test followed by Dunn's multiple comparisons post test). (B) Local and systemic IL-28 production in mice after cecal ligation puncture (CLP)–induced sepsis. C57BL/6 mice (n = 6 per group) were subjected to sham or CLP. Organs were removed at the indicated time points, blood was collected by cardiac puncture, and peritoneal lavage ﬂuid (PLF) was obtained by washing the peritoneal cavity with 5 ml of sterile PBS. Samples were assayed for IL-28 content by ELISA. *p < .05, **p < .01,***p < .001, compared with sham control mice (Mann–Whitney U test).

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2.7. Determination of bacterial load

Serial dilutions of blood and peritoneal lavage ﬂuid (PLF) were prepared in sterile PBS for plating on brain-heart-infusion agar plates. Colony- forming unit (CFU) counts were then determined after 24-hour culture.

2.8. Statistical analysis

Human data were expressed as scatter dot plots with medians. Mice data were expressed as box-and-whisker plots showing the smallest observation, lower quartile, median, upper quartile, and largest observation or as medians with interquartile ranges. Comparisons between groups were tested using the Mann-Whitney U test or Kruskal- Wallis test followed by Dunn's multiple comparisons post test as ap- propriate. For survival studies, Kaplan-Meier analyses followed by log- rank tests were performed. All analyses were done using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA). p values < .05 were considered statistically signiﬁcant.

Fig. 2. Neutralization of IL-28 activity with monoclonal antibody attenuated 3. Results CLP-induced sepsis. C57BL/6 mice were subjected to CLP, 10 μg of anti-mouse IL-28A/B neutralizing monoclonal antibody was administered intraperitoneally 3.1. The levels of IL-28 were up-regulated in human and murine sepsis in 50 μl of PBS at 2 h after CLP, followed by a booster dose of 10 μg at 8 h later after CLP. As a control, rat IgG2b control antibody was used. (A) Survival of To ascertain the relevance of IL-28 in human sepsis, we examined septic mice (n = 15 per group) following IL-28 neutralization after CLP with IL-28A (IFN-λ2) levels from the blood of septic patients and healthy anti-IL-28 antibody. Comparison between groups was done by Kaplan–Meier controls (Table 1). Serum IL-28A levels were significantly elevated in analysis followed by log-rank tests. *p < .05 when compared with septic mice treated with isotypical IgG control. Results are representative of three in- patients with sepsis compared to healthy controls, and those who did fi dependent experiments. (B) Dilutions of PLF and blood obtained from septic not survive displayed signi cantly more serum IL-28A than did the mice (n = 6) at 48 h after CLP were cultured on blood agar plates, and the survivors (Fig. 1A). We also analyzed the production of IL-28 in the number of bacterial colonies was counted as CFU. *p < .05 when compared murine model of CLP-induced sepsis, and found that IL-28 concentra- with CLP-induced septic mice treated with isotypical IgG control tions were significantly increased in the PLF, blood and lung at 24 or (Mann–Whitney U test). 48 h after CLP (Fig. 1B).

2.4. In vivo blockade of IL-28 3.2. Antibody against IL-28 protected against lethal sepsis

To block IL-28 during experimental sepsis, we used rat anti-mouse Having established that IL-28 release was up-regulated in clinical IL-28A/B neutralizing monoclonal antibody (R&D systems, monoclonal and experimental sepsis, we next studied the role of IL-28 in CLP-in- rat IgG2b Clone # 244716). In vitro studies have shown that reduced sepsis. We firstly used an IL-28A/B blocking mouse monoclonal combinant mouse IL-28B reduced the Encephalomyocarditis Virus antibody (Clone # 244716) to neutralize the biologic activity of IL-28. (EMCV)-induced cytopathy in the HepG2 human hepatocellular carci- Using CLP-induced sepsis model, we observed that the survival rate in noma cell line, and inhibition of EMCV activity elicited by recombinant mice treated with anti-IL-28A/B antibody was significantly higher than mouse IL-28B could be neutralized by this antibody. The 50% neu- that in mice treated with immunoglobulin G (IgG) control (Fig. 2A). tralizing dose (ND50) is typically 3–9 μg/mL. In mouse studies, 10 μgof Furthermore, mice treated with anti-IL-28A/B antibody displayed a anti-mouse IL-28A/B neutralizing monoclonal antibody was adminis- significant decrease in bacterial loads from peritoneum and blood at tered intraperitoneally in 50 μl of PBS 2 h after CLP, followed by a 48 h after CLP (Fig. 2B). booster dose of 10 μg 8 h later after CLP. As a control, rat IgG2b control antibody was used. 3.3. Treatment with anti-IL-28 antibody enhanced neutrophil infiltration into the peritoneum during sepsis

2.5. In vivo administration of IL-28 Because leukocytes are critical for host defense during sepsis, we investigated whether IL-28 modulates leukocyte influx in sepsis. There Recombinant murine IL-28A or IL-28B protein (2 μg, R&D systems) were significantly greater numbers of leukocytes in PLF from anti-IL- was injected immediately after CLP or S. aureus infection. PBS was 28A/B-treated mice compared with IgG-treated mice (Fig. 3A and B). delivered in a similar fashion as control vehicle. Moreover, we found that the number of neutrophils but not macrophages and lymphocytes was significantly increased with IL-28A/B blockade (Fig. 3A and B). 2.6. Differential cell counts in peritoneum 3.4. IL-28A delivery exacerbated lethality during sepsis Peritoneal lavage was performed with 4 mL of PBS containing 1 mM ethylenediaminetetraacetic acid. Peritoneal cell suspension was pel- Because the administration of antibody against IL-28 was found to leted and resuspended. Cell viability was determined using Trypan blue protect mice from lethal sepsis, we performed the reverse experiment exclusion assay, and cell numbers were counted with a hematology and examined the effect of a bolus injection of recombinant mouse IL- analyzer. Cytospin slides were prepared and stained with a Wright- 28 given at the onset of CLP. IL-28A supplementation in the absence of Giemsa stain. sepsis had no effect on survival in healthy mice (Fig. 4A). In this model of CLP-induced sepsis, recombinant IL-28A significantly decreased

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Fig. 3. Neutralization of IL-28 activity with monoclonal antibody enhanced neutrophil inﬁltration during CLP-induced sepsis. (A) Cytospin centrifugation was performed for Diﬀ-Quik staining (x 40) to assess cell counts in PLF from septic mice (n = 6) with or without IL-28 neutralization at 24 h after CLP. (B) Number of cells in PLF from mice (n = 6) with or without IL-28 neutralization at 24 h after severe CLP. ***p < .001 when compared with septic mice treated with isotypical IgG control (Mann–Whitney U test).

Fig. 4. Treatment with recombinant IL-28A aggravated experimental sepsis. (A) Survival of septic mice (n = 15 per group) following IL-28A supplementation. Recombinant murine IL-28A (2 μg/injection) was injected at the time of CLP. PBS was delivered in a similar fashion as control vehicle. Comparison between groups was done by Kaplan–Meier analysis followed by log-rank tests. *p < .05 when compared with septic mice treated with PBS control. Results are representative of three independent experiments. (B) Dilutions of PLF and blood obtained from septic mice (n = 6) at 48 h after CLP were cultured on blood agar plates, and the number of bacterial colonies was counted as CFU. *p < .05 when compared with CLP-induced septic mice treated with isotypical IgG control (Mann–Whitney U test).

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produced substantial survival benefit in experimental sepsis; (3) IL-28 supplementation in the presence of sepsis worsened outcome of experimental sepsis. Since its discovery in 2003, many groups have identified contribu- tions of IL-28 to host immune responses. IL-28 production has been found to be increased in serum from patients with chronic hepatitis C virus (HCV) infection or dengue virus infection [12,13]. Increased serum levels of IL-28 were also found in patients with Hashimoto's Thyroiditis [14]. This is the first study to analyze IL-28 levels in blood samples from patients with sepsis. Serum IL-28 levels were elevated in patients with sepsis compared to healthy donors. Notably, those who did not survive had significantly more serum IL-28 than did the survivors. However, we are unable to determine whether this is a biomarker for illness severity of human sepsis, and we acknowledge this can only be definitively established in humans with a large cohort of clinical study. Our data suggest a novel role of IL-28 in the progression of experimental sepsis. Neutralization of the IL-28 activity with antibody fi Fig. 5. Treatment with recombinant IL-28A restricted neutrophil infiltration against IL-28 could attenuate CLP-induced sepsis. The survival bene t during CLP-induced sepsis. Recombinant murine IL-28A (2 μg/injection) was obtained with neutralizing antibody against IL-28 was associated with injected at the time of CLP. PBS was delivered in a similar fashion as control lower local and circulating bacterial counts. This finding is in agree- vehicle. Number of cells in PLF from septic mice (n = 6) treated with or without ment with the recent observation that activation of IL-28 signaling in recombinant IL-28A at 24 h after CLP. ***p < .001 when compared with septic response to influenza virus infection increased susceptibility to pul- mice treated with PBS control (Mann–Whitney U test). monary infection by methicillin-resistant Staphylococcus aureus (MRSA) [15]. The improved bacterial clearance obtained with IL-28 neutralization was associated with enhanced neutrophil infiltration during CLP. A previous study has demonstrated that treatment with recombinant IL-28A restricted neutrophil infiltration into joint in the mouse model of collagen-induced arthritis [6]. Here we also confirmed that IL-28 supplementation in septic mice suppressed neutrophil influx during CLP. Taken together, these results suggest that IL-28 might im- pair bacterial clearance during sepsis by restricting neutrophil influx to the site of infection. Collectively, our data suggest that IL-28 plays a detrimental role in the pathogenesis of sepsis. Elevated IL-28 production during sepsis may promote bacterial dissimilation by limiting neutrophil filtration, Fig. 6. Treatment with recombinant IL-28B aggravated mortality during CLP- leading to a failure to contain the infection, thereby resulting in the induced sepsis. Survival of septic mice (n = 15 per group) following IL-28B aggravation of sepsis. Therefore, blocking IL-28 may offer a new supplementation. Recombinant murine IL-28B (2 μg/injection) was injected at strategy for the future of immune therapy in sepsis. the time of CLP. PBS was delivered in a similar fashion as control vehicle. – Comparison between groups was done by Kaplan Meier analysis followed by Acknowledgments log-rank tests. *p < .05 when compared with septic mice treated with PBS control. Results are representative of three independent experiments. This study is supported by National Natural Science Foundation of China grants (No. 81572038 and No. 81722001 to J.C.), Chongqing survival of septic mice compared to PBS control (Fig. 4A). Treatment Science and Technology Commission Grant for Distinguished Young with recombinant IL-28A also impaired bacterial clearance in the Scholars of Chongqing (cstc2014jcyjjq10002 to J. C.) peritoneum and blood (Fig. 4B). Furthermore, there were significantly lower numbers of leukocytes in PLF from IL-28A-treated septic mice Conflicts of interests compared with PBS-treated septic mice, and the number of neutrophils but not macrophages and neutrophils was reduced with IL-28A treat- The authors declare that there is no conflict of interests regarding ment in septic mice (Fig. 5). the publication of this paper.

3.5. Treatment with IL-28B exacerbated lethality during sepsis References

To further confirm the detrimental effects of IL-28 on sepsis, re- [1] T. van der Poll, F.L. van de Veerdonk, B.P. Scicluna, M.G. Netea, The im- combinant IL-28B (IL-28A and -B are 96% identical [11]) was instilled munopathology of sepsis and potential therapeutic targets, Nat. Rev. Immunol. 17 – into septic mice. Addition of recombinant IL-28B (2 μg) to septic mice (2017) 407 420. [2] M.P. Fink, H.S. Warren, Strategies to improve drug development for sepsis, Nat. also increased CLP-induced lethality (Fig. 6). Rev. Drug Discov. 13 (2014) 741–758. [3] L.A. van Vught, M.A. Wiewel, A.J. Hoogendijk, J.F. Frencken, B.P. Scicluna, P.M.C. Klein Klouwenberg, A.H. Zwinderman, R. Lutter, J. Horn, M.J. Schultz, 4. Discussion M.M.J. Bonten, O.L. Cremer, T. van der Poll, The host response in patients with sepsis developing intensive care unit-acquired secondary infections, Am. J. Respir. In this study, we identified IL-28 to be centrally involved in the Crit. Care Med. 196 (2017) 458–470. λ pathogenesis of sepsis. We made the following key observations: (1) [4] H.M. Lazear, T.J. Nice, M.S. Diamond, Interferon- : immune functions at barrier surfaces and beyond, Immunity 43 (2015) 15–28. patients with sepsis have increased circulating IL-28 levels, and IL-28 [5] C. Sommereyns, S. Paul, P. Staeheli, T. Michiels, IFN-lambda (IFN-lambda) is ex- release was enhanced in the experimental model of sepsis; (2) pressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo, Neutralization of the IL-28 activity with antibody against IL-28 PLoS Pathog. 4 (2008) e1000017.

33 Q. Luo, et al. Clinical Immunology 205 (2019) 29–34

[6] K. Blazek, H.L. Eames, M. Weiss, A.J. Byrne, D. Perocheau, J.E. Pease, S. Doyle, Infect. Dis. 215 (2017) 321–332. F. McCann, R.O. Williams, I.A. Udalova, IFN-λ resolves inflammation via suppres- [11] P. Sheppard, W. Kindsvogel, W. Xu, et al., IL-28, IL-29 and their class II cytokine sion of neutrophil infiltration and IL-1β production, J. Exp. Med. 212 (2015) receptor IL-28R, Nat. Immunol. 4 (2003) 63–68. 845–853. [12] H. Park, E. Serti, O. Eke, K. Henderson, S. Schlutsmeyer, T.E. Whitmore, [7] O. Koltsida, M. Hausding, A. Stavropoulos, S. Koch, G. Tzelepis, C. Ubel, R. Kuestner, U. Garrigues, C. Birks, J. Roraback, C. Ostrander, D. Dong, J. Shin, S.V. Kotenko, P. Sideras, H.A. Lehr, M. Tepe, K.M. Klucher, S.E. Doyle, S. Presnell, B. Fox, B. Haldeman, E. Cooper, D. Taft, T. Gilbert, F.J. Grant, M.F. Neurath, S. Finotto, E. Andreakos, IL-28A (IFN-λ2) modulates lung DC func- M. Tackett, W. Krivan, G. McKnight, C. Clegg, D. Foster, K.M. Klucher, IL-29 is the tion to promote Th1 immune skewing and suppress allergic airway disease, EMBO. dominant type III interferon produced by hepatocytes during acute hepatitis C virus Mol. Med. 3 (2011) 348–361. infection, Hepatology 56 (2012) 2060–2070. [8] M. Singer, C.S. Deutschman, C.W. Seymour, M. Shankar-Hari, D. Annane, M. Bauer, [13] C.H. Hung, C.H. Huang, L. Wang, C.C. Huang, M.C. Wu, Y.Y. Chin, C.Y. Lin, R. Bellomo, G.R. Bernard, J.D. Chiche, C.M. Coopersmith, R.S. Hotchkiss, K. Chang, D.C. Wu, Y.H. Chen, IL-28 and IL-29 as protective markers in subject with M.M. Levy, J.C. Marshall, G.S. Martin, S.M. Opal, G.D. Rubenfeld, T. van der Poll, dengue fever, Med. Microbiol. Immunol. 206 (2017) 217–223. J.L. Vincent, D.C. Angus, The third international consensus definitions for sepsis [14] D. Arpaci, S. Karakas Celik, M. Can, G. Cakmak Genc, F. Kuzu, M. Unal, and septic shock (Sepsis-3), JAMA 315 (2016) 801–810. T. Bayraktaroglu, Increased Serum Levels of IL-28 and IL-29 and the Protective [9] Z. Song, X. Zhang, L. Zhang, F. Xu, X. Tao, H. Zhang, X. Lin, L. Kang, Y. Xiang, Effect of IL28B rs8099917 Polymorphism in Patients with Hashimoto's Thyroiditis, X. Lai, Q. Zhang, K. Huang, Y. Dai, Y. Yin, J. Cao, Progranulin plays a central role in Immunol. Investig. 45 (2016) 668–678. host defense during sepsis by promoting macrophage recruitment, Am. J. Respir. [15] P.J. Planet, D. Parker, T.S. Cohen, H. Smith, J.D. Leon, C. Ryan, T.J. Hammer, Crit. Care Med. 194 (2016) 1219–1232. N. Fierer, E.I. Chen, A.S. Prince, Lambda interferon restructures the nasal micro- [10] X. Tao, Z. Song, C. Wang, H. Luo, Q. Luo, X. Lin, L. Zhang, Y. Yin, J. Cao, Interleukin biome and increases susceptibility to Staphylococcus aureus superinfection, MBio 7 36α Attenuates Sepsis by Enhancing Antibacterial Functions of Macrophages, J. (2016) (e01939-15).